The long range goal of this research is to prepare site-specific covalent adducts that represent the products formed when various carcinogens react with DNA in vivo and to examine the mutagenic effect of these lesions both in vivo and in vitro in order to define the precise series of biochemical events that leads to mutations. Ultimately we would like to know whether this series of events is prerequisite to tumor production. Our initial objective is to answer the following specific questions: (1) Which of the covalent adducts produced in DNA by reactions with different carcinogens are mutagenic? (2) What type of mutation (base substitution, frameshift, large deletion or rearrangement) does each kind of DNA adduct produce? (3) What roles do various DNA repair systems play in producing these mutations? (4) Is there any correlation between a particular type of DNA adduct produced by known carcinogens and the carcinogenic index of that carcinogen. We have developed a system using bacteriophage phiX174 that we believe is capable of answering these questions. We are introducing different potentially mutagenic lesions into single preselected sites in gene G (an essential gene) by a combination of chemical and enzymatic syntheses. The expression of these site-specific modifications is being examined in vivo by transfection of spheroplasts derived from various E. Coli mutants that are defective in certain DNA repair systems. Mutant virus present in the spheroplast lystae is detected and propagated using a phiX-sensitive host carrying a functional copy of phiX gene G on a plasmid. This phiXG system is permissive for all types of mutation in gene G. Viral DNA carrying mutations is isolated from the rescued phage and sequenced in the region of interest to define the precise nature of the mutations that have occurred in response to the original site-specific covalent modification.